Protocol for chromatin immunoprecipitation of histone modifications in frozen adipose tissue

Summary Chromatin immunoprecipitation (ChIP) combined with sequencing has revolutionized our understanding of gene regulation; however, its application to frozen adipose tissue presents unique challenges due to the high levels of lipid content. Here, we present a protocol for ChIP of histone modifications in human frozen adipose tissue. We describe steps for tissue preparation, chromatin isolation, sonication, pre-clearing of chromatin, and immunoprecipitation. We then detail procedures for elution, crosslink reversal, chromatin purification, quality control, and library synthesis.


MATERIALS AND EQUIPMENT Buffers
Alternatives: All the reagents used in this protocol can be replaced with the same function from different suppliers.Nuclei lysis with SDS Prepare immediately before the experiment and store it on ice.

Nuclei lysis without SDS
Prepare immediately before the experiment and store it on ice.

Elution buffer
Prepare freshly.

Wash buffer 1
Store at 4 C for up to 12 months.

Reagents Final concentration Amount
Tris pH 8. Alternatives: Any magnetic rack is suitable for magnetic separation Omni Tissue Homogenizer.
Alternatives: Any homogenizer to disrupt the soft tissues such as Benchmark Scientific D1000 Handheld Homogenizer Bioruptor Plus Diagenode Sonicator.
Alternatives: Any rotator with adjustable time and rotating speed VWR centrifuge with a swing-out rotor for 1.5-mL tubes.
Alternatives: Any centrifuge is suitable for 1.5 mL tubes with cooling system Thermo Fisher Scientific Centrifuge with a swing-out rotor suited for 15-mL conical tubes.
Alternatives: Any centrifuge is suitable for 1.5 mL tubes with cooling system IKA Vortex.
Alternatives: Any mixer with controlling temperature and time BIO-RAD Agarose gel electrophoresis apparatus.
Alternatives: Any gel system Thermo Fisher Scientific Qubit spectrophotometer.
Alternatives: Any qPCR machine

STEP-BY-STEP METHOD DETAILS
Preparation of adipose tissues for the fixation

Timing: 120 min
This protocol is designed for a single ChIP assay targeting H3K36me3.It has been optimized for different human frozen adipose tissue depots, specifically SAT and OVAT, which were snap frozen and stored at À80 C post-surgery.In this step, we prepare frozen adipose tissues for the fixation.
CRITICAL: Cut the tissue in a mortar using a pre-chilled scalpel on dry ice.

Processes
1. Take the samples from À80 C into dry ice.
2. Put tissue samples in the pre-cooled mortar with dry ice.a. Place sarstedt screw cap tubes (15 mL) are in dry ice.b.By using a cold scalpel, cut tissue samples into smaller pieces ($2-3 mm). 3. Add 3 mL of 1% FA (fresh) in PBS (Ca/Mg free) to each tube containing approximately 100 mg adipose tissue.a.Put on slight rotation at 30 rpm at room temperature for 10 min, avoiding from direct light.
CRITICAL: Weight adipose tissues just before experiment.
4. Cross-linking is stopped by adding 158 mL of Glycine (2.5 M) in PBS.

Note:
The final concentration of Glycine is 0.125 M.
5. Continue rotating at room temperature for 10 min at 30 rpm at room temperature.6. Centrifuge the tubes at 470 g at 4 C for 10 min at COLD ROOM (+4 C). 7. Following centrifugation, remove gently the liquid phase by a glass pipette.
Note: There may be two layers of tissue observed either settling at the bottom of the tube or forming a top layer.Some samples may exhibit tissue cuts appearing to float within the liquid.The primary objective here is to eliminate the liquid phase containing FA and Glycine while ensuring that small tissue fragments remain untouched.It is essential to maintain the samples on ice throughout this process.
8. Wash the samples with the resulting tissue pellet and the tissue from the top layer three times with 5 mL of cold PBS with PIC (20 mL per 1 mL).9. Centrifuge tubes at 470 g at 4 C for 10 min.
a.The liquid phase is removed by a glass pipette.
Note: Work in the hood.

Chromatin isolation and sonication
Timing: 240 min Here, we isolate chromatin and apply sonication process for the chromatin fragmentation.
Note: Add PIC, NaBu, and PMSF just before use.
Note: Maintain all buffers and samples on ice during this stage.
Note: Prepare nucleus lysis buffer, with and without SDS, which we used to adjust the final SDS concentration to 0.5%.
10. Add 5 mL of cold cell lysis buffer with PIC, NaBu, and PMSF to each tube from step 9 (on ice).11.Homogenize the tissue by an OMNI homogenizer with Hard Tissue Omni Tip (blue) Note: 3 times 10 sec with a 30-sec break on ice in between.
12. Incubate tubes on ice for 30 min while vortexing (10 s) every 3 min.13.After incubation, centrifuge at 2870 g for 10 min at 4 C to precipitate the resulting cell pellet.14.Remove the supernatant by inverting the tube.

Note:
The tube can be left upside down on paper to avoid lipids running down onto the pellet.
15. Add 1 mL of cell lysis buffer with PIC, NaBu, and PMSF to each tube.Mix by vortexing and centrifuge at 2,870 g for 10 min at 4 C Note: This process enables to precipitate the core fraction, which can help to remove excess fat.
16. Re-suspend the nuclear pellet in 150 mL cold nucleus lysis buffer (plus 1.0% SDS) with PIC, NaBu, and PMSF and transfer to a 'sonication' tube (1.5 mL).17.Vortex and incubate the tubes for 2 h on ice.18.After 2 h, add 150 mL cold nucleus lysis buffer without SDS.
Note: This is required for shearing chromatin at the optimal size range.Thus, the final SDS concentration is 0.5%.The total volume of the chromatin solution is 300 mL.
Note: Use the following settings: High power, 30 s ON/ 30 s OFF, 8 cycles, repeat 4 times; 32 cycles in total.After each cycle, briefly, vortex, spin and incubate for 2 min on ice (Figure 1).20.After shearing, centrifuge tubes at 10,000 g at 4 C for 15 min.
Note: Following centrifugation, the tube exhibits three distinct layers.The bottom layer comprises tissue debris, while the top layer appears blurry and is thinner.Positioned between these layers is a transparent layer containing chromatin.
21. Collect transparent layer without disturbing the bottom and top layers and transfer it to DNA low-binding tubes (1.5 mL).22.Take 10 mL of chromatin for quality and quantity control.

Timing: Overnight
Here, we pre-clear the chromatin by incubating it with the beads.
23.A pre-clearing step can be performed here before storing the chromatin at À80 C.
Note: This is optional and pre-clearing of the chromatin can also be performed directly before immunoprecipitation.
24. Wash 10 mL of protein-G dynabeads with 100 mL of RIPA Buffer containing 0.1% SDS three times.25.Allow a 2 min incubation period on ice, briefly spin, place the tube on a magnetic plate, and then remove the buffer by pipetting.26.Following the last wash, without resuspending in any buffer again, a. Add isolated chromatin (100 mL) immediately to protein G dynabeads beads (10 mL) within 1.5 mL low-binding DNA tubes b.Rotate overnight at COLD ROOM (+4 C). 27.Perform a quick spin to collect the liquid that is trapped in the cap.
a. Place the tube containing chromatin and beads mix in a magnetic rack to hold 1.5 mL tubes.b.Wait for 2 min.c.Take the buffer containing pre-cleared chromatin.d.Put it in a new 1.5 mL low-binding DNA tube Note: Place magnetic plate in ice during pre-clearing processes 28.Store pre-cleared chromatin at À80 C without snap freeze.

Timing: 105 min
Here, we prepare the chromatin to determine the quality and quantity.
29.Take 10 mL of the pre-cleared chromatin from step 28.a. Add 1 mL RNase A (from 500 ng/mL stock).b.Incubate at 37 C for 20 min at 500 rpm using Thermomixer.30.Prepare elution buffer and add 190 mL of fresh elution buffer (add 1% SDS before use).31.Add 5 mL proteinase K from a 10-fold dilution of the 20 mg/mL stock and incubate at 37 C for 20 min.32.Incubate at 68 C at 500 rpm for 1 h.

Purification and analysis of chromatin fragment size
Timing: 90 min Here, we isolate chromatin and determine the fragment size.Alternatives: 2100 Bioanalyzer system (Agilent, USA) is an alternative approach for the fragment size and distribution.Briefly, prepare the gel-dye mix by high sensitivity DNA dye with high sensitivity DNA gel matrix.Load 9 mL of the gel-dye mix in wells in high sensitivity DNA chip as indicated in the protocol.Load 5 mL of green-capped high sensitivity DNA marker into the wells marked with the ladder symbol.Load the 1 mL of ladder and the samples, put in vortex mixer and start the chip run.
Alternatives: Nanodrop offers an alternative approach, consistently providing concentration measurements that are higher than those obtained with the Qubit due to a lack of sensitivity.Qubit provides high sensitivity, whilst Nanodrop provides purity measurements.

Immunoprecipitation step
Coupling of dynabeads with histone modification-specific antibody

Timing: 135 min
We incubate dynabeads with histone modification-specific antibody for coupling.
43. Vortex the bottle of dynabeads stock for 1 min before taking out the beads.44.Take 10 mL of beads per ChIP in a 1.5 mL tube (low DNA binding).

Note:
We test that the incubation duration differ for various antibodies targeting H3K4me3, H3K4me1, H3K27me3, and H3K27ac.For those modifications, we investigate the effects of 0.5, 1, and 2 h incubation periods and subsequently compare the immunoprecipitation percentages obtained from each duration.

Timing: 135 min
Here, we immunoprecipitate the chromatin.
51.After 2 h of incubation, a.Put the tube on the magnet for 1 min.b.Remove the buffer containing the unconjugated antibody.52.Adding pre-cleared chromatin ($500 ng DNA in 100 mL) and in parallel; a.Put 5% of chromatin as INPUT in another DNA low-binding tube.b.Place the input samples on ice until being processed in step 64.

Note:
The initial concentration of chromatin primarily determines the incubation time.Using $500 ng of DNA in total, necessitates a 2-h incubation with antibody-bead complex.
Note: The final chromatin volume must be 100 mL.Using a larger volume might lead to increased non-specific binding.
Note: If the DNA concentration is elevated, adjust both the volume and concentration using nucleus lysis buffer (with 0.5% SDS).
Note: Conversely, if the DNA concentration is lower, ensure that up to 300 ng of DNA in total per ChIP can be utilized.
Note: As per the protocol recommendation, when working with 500 ng of DNA, employ 25 mL to achieve a 5% proportion for input.
53. Incubate the tubes horizontally (pre-cleared chromatin + beads-antibody complex) on a rotator at 40 rpm in the cold room (+4 C) for 2 h.

Washing of the immunoprecipitated material
Timing: 90 min We wash the immunoprecipitated material to remove unspecific chromatin or debris.
54.After incubation, snap-spin the tubes in a micro-centrifuge to bring down any solution trapped in the lid.55.Place the tubes on a magnet rack on ice for 1 min.56.Remove the supernatant while tubes are on the magnet.
a. Throw away the supernatant.
Note: Be careful to avoid the dynabeads.Note: Prepare all buffers freshly and keep on ice.

Timing: Overnight
We elute and de-crosslink the CHIP material from the beads.
63. Prepare elution buffer (fresh-room temperature) and add 141.25 mL of an elution buffer containing 1% SDS and 5 mL of RNase (500 ng/mL) to ChIP.64.Add 282.5 mL of an elution buffer containing 1% SDS and add 10 mL of RNase (500 ng/mL) in INPUT (5% of chromatin).65.Incubate ChIP and INPUT samples at 37 C, 1300 rpm rotating for 20 min using Thermomixer.66. Add 3.75 mL and 7.5 mL of proteinase K (2 mg/mL) to ChIP and INPUT, respectively and incubate at 37 C, 1300 rpm for 20 min.67.Incubate ChIP and INPUT at 68 C overnight at 1300 rpm on a Thermomixer for reverse cross linking.68.After overnight incubation, snap-spin samples-beads mix, and place on the magnet for 1 min, and transfer the buffer into 2 mL low binding DNA tube.69.Add 150 mL of elution buffer (fresh-room temperature) containing 1% SDS to the beads again and incubate at 68 C for 10 min at 1300 rpm on a Thermomixer.

Timing: 180 min
We run RT-qPCR to control the enrichment in positive and negative control genomic regions.
To assess the success of the ChIP protocol, we recommend employing a target-specific enrichment approach via RT-qPCR.This involves enriching a specific genomic region with H3K36me3, serving as a positive control for an active genomic region (Table 1), and another specific region serving as a negative control (Table 2).For the positive control, we propose a genomic region in the Actin gene and ADIPOQ gene, while a primer set from Active Motif (Human negative control primer set 1) is utilized for the negative control, targeting a gene desert on chromosome 12 (Figure 2).We perform triplicate RT-qPCR runs for each sample and input and then calculate the mean Ct value per sample.To determine chromatin enrichment (% input), we initially apply a logarithmic transformation (log2) to 20-fold (100/5) as it is calculated as 4,321928.Then, we calculate adjusted 100% IP = (Input) -4,321928.Subsequently, we compute delta Ct as the difference between Ct values of the adjusted 100% IP and the samples.Finally, we calculate the percentage of input (% input) using the formula: 100 * 2^(Delta Ct)."

Sequencing library preparation
Timing: 120 min In this section, we perform library synthesis.
Utilizing ChIP/INPUT materials from samples, we generate a sequencing library for subsequent sequencing, constituting a pool of DNA fragments with specific adapters designed for sequencing.We adhere to the guidelines provided by a commercial kit (Takara Bio USA ThruPLEX DNA-Seq).The library preparation involves three main steps: template preparation, library synthesis, and library amplification.While we do not delve into all details of these steps, which are available in the kit's guideline, we emphasize on several critical considerations for a successful library preparation: Note: Thermal cycler conditions, including a heated lid set to 101 C-105 C and ramp rates ranging from 3 C/s to 5 C/s.

Note:
The amount of ChIP material in TE buffer as an input buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0).We utilize 0.20 ng of total ChIP/INPUT material for library preparation.

Note:
The cycle number in the library amplification step.We employ 15 cycles for this stage.
For the sequencing library preparation, we employ the DNA Unique Dual Index Kit (Takara/AH Diagnostics), incorporating index primers carrying the 8-nt long index sequences "IDT for Illumina TruSeq DNA."It's noteworthy that the kit recommends using input DNA in the range of 50 pg to 50 ng as starting material.We successfully perform library synthesis using 0.20 ng of total ChIP/INPUT material in a 10 mL final volume.Here, we remove small fragments less than 100 bp.
Following library preparation, we employ Agencourt AMPure XP beads (Beckman Coulter, USA), known for selectively binding to PCR amplicons with a size of 100 bp and larger.By following the protocol (000387v001), we successfully a sequencing library containing amplicons exceeding 100 bp.To assess the library's quality, we utilize the 2100 Bioanalyzer system (Agilent Technology, USA) with a high sensitivity DNA kit.The electropherograms obtained for two adipose tissue samples and their INPUTs indicate that the library's size is optimal for sequencing (Figure 4).

EXPECTED OUTCOMES
Our specific protocol evolves through modifications of key steps from established ChIP protocols.
Given the diverse cellular composition and structure inherent in adipose tissues, our protocol undergoes optimization to ultimately achieve high-quality sequencing data for various histone modifications in both tissue depots concurrently.

Chromatin concentration after isolation process
The established protocol utilizes a minimal quantity of frozen human adipose tissues.According to our protocol, approximately 100 mg of adipose tissue is anticipated to produce around 2000-2500 ng of chromatin in total.It's noteworthy that we observe a higher chromatin concentration in OVAT samples (13.43 G 8.26 ng/mL, n = 12) compared to SAT samples (8.81 G 5.84 ng/mL, n = 12).In summary, using 100 mg of either SAT or OVAT should provide sufficient chromatin material for the ChIP process.

Sonication
We anticipate obtaining high-quality and properly fragmented chromatin from human adipose tissue.As we observe, there is a notable variation between samples originating from different depots, particularly among intra-individually paired samples.Consequently, we foresee the protocol's capability to generate appropriately fragmented chromatin (mainly ranging between 200-600 bp) for all individuals.Through extensive testing under various conditions, we determine that 8 cycles, repeated 4 times (32 cycles), result in optimal chromatin fragmentation (Figure 1).Following each set of 8 cycles, the samples are mixed and kept on ice for 2 min before proceeding with the subsequent cycle.It's worth noting that OVAT tissue samples may exhibit variations in response to sonication conditions, potentially necessitating additional sonication.

Chromatin immunoprecipitation
We systematically test various conditions, including different bead types, antibodies, and incubation times for beads and antibodies, as well as chromatin, to eliminate non-specific binding while achieving high-quality enrichment of the positive genomic region.The protocol is validated for 400-500 ng of chromatin.While theoretically, 25 mg of tissue might suffice for the ChIP process, we recommend starting with 100 mg of frozen adipose tissue.Despite the protocol requiring a relatively low concentration of chromatin when compared with previous protocol, 2,3 we anticipate achieving higher enrichment of the positive control (Figures 2 and 3).In summary, the ChIP/Input chromatin obtained for each sample is sufficient for q-PCR analysis and subsequent library preparation.

Expected RT-qPCR results
Prior to sequencing, it's imperative to validate the protocol's efficacy.Hence, RT-qPCR serves as a critical checkpoint to ascertain its success.To evaluate the effectiveness of each procedural step, we select two genomic regions: one rich in H3K36me3 and a commercial negative control.We assess enrichment via RT-qPCR to gauge the coherence of the process, calculating IP% based on the obtained data.Ideally, we anticipate negligible enrichment (around 1% or less) for the negative control and substantial enrichment for H3K36me3.Elevated enrichment in the negative control would suggest non-specific binding. in case of H3K36me3, obtaining$ 10% IP or more in the positive region indicates a successful enrichment.In our experiment, employing the refined protocol on human SAT and OVAT samples from 12 subjects, we observe higher enrichment in the negative control, while achieving the desired enrichment in the positive region, paving the way for sequencing."

Quality of library
We expect to obtain high quality of library.We obtain libraries with high quality; proper chromatin fragment sizes and sufficient quantity for sequencing after preparing 12 samples of SAT and OVAT (Figure 4).

LIMITATIONS
This protocol is designed with the following objectives: 1) to achieve a higher chromatin yield, 2) to conduct successful immunoprecipitation with minimal nonspecific background, 3) to enable successful high quality library synthesis, and 4) to produce high-quality sequencing data for H3K36me3.We also apply the protocol's applicability to H3K4me3, H3K4me1, H3K27me3, and H3K27ac modifications by testing various antibody incubation times with beads and the antibody-beads complex with chromatin.We obtain successful results.While each step of the protocol undergoes thorough testing, it's important to note that when targeting different modifications or proteins, critical parameters include antibody type, incubation with beads, and chromatin interaction.These parameters are modification-specific and require precise adjustments.
Notably, the protocol is not validated for cells from cell culture (e.g., pre-adipocytes or mature adipocytes) or non-adipose tissues.Therefore, optimization for specific cell types or tissues may be necessary.

TROUBLESHOOTING
The primary goal of this protocol is to ensure successful immunoprecipitation, library synthesis, and the generation of high-quality data.Successful tests are conducted for H3K36me3 histone modification.More importantly, the protocol shows successfully application for additional four distinct histone modifications in human SAT and OVAT samples.

Problem 1
Variation in Chromatin Amount.
It expects due to variations in chromatin quantity among individual samples in a population study, especially with some samples exhibiting lower concentrations.

Potential solution
The lowest concentration obtained in our study (n = 12) is sufficient for ChIP and INPUT.For potential experiment repeats, we suggest the use of over 100 mg of tissue for samples with low chromatin amounts.

Problem 2
Sonication Efficiency in OVAT Samples.
Varying efficiency in sonication, leading to varied fragment sizes in OVAT samples.

Potential solutions
After testing different conditions, an optimal solution is applying 5 3 8 = 40 cycles of sonication instead of 4 3 8 = 32 cycles to achieve the proper size.The use of Covaris ultrasonicator can be beneficial, with testing required for each Covaris conditions to eliminate variations.

Problem 3
Type of Antibody and Concentration.

Figure 1 .
Figure 1.An example of different sonication conditions (Cy: cycle) 33. Purify the chromatin withMinElute PCR purification kit.34.Add 5 volumes (1025 mL) of binding buffer (PB) to the sample.a. Add 40 mL of 3 M NaAc (pH: 5.2).35.Add 700 mL of the sample in MinElute column.a. Centrifuge 1 min at 13,000 rpm.b.Remove flow through.36.Load the rest of the samples in the same column until loading all the samples.a. Centrifuge 1 min at 13,000 rpm.b.Remove flow through.37. Wash with 700 mL of PE buffer.a. Centrifuge 1 min at 13,000 rpm.b.Remove flow through.38.Put the samples in a new collection tube and centrifuge for 1 min at 13,000 rpm.39.Put MinElute column in 1.5 mL low-DNA binding tube.a. Add 10 mL TE (Tris-EDTA) Buffer (pH = 8.0).b.Incubate for 1 min at 37 C. c. Centrifuge 1 min at 13,000 rpm.40.Maintain the samples at 55 C for 5-10 min to allow ethanol evaporation beforehand.Note: To remove any residual alcohol from the reaction.41.Use Qubit dsDNA HS kit and determine the dsDNA concentration by Qubit fluorometer.a. Prepare the standards used in the calibration.b.Prepare working solution.c.Add 1 mL of DNA to working solution.d.Measure the concentration.42.Add 1 mL gel-pilot loading (53, QIAGEN) dye + 5 mL of chromatin (200-250 ng in total).a. Run at 90-100 V (45-60 min).b.Analyze DNA in a 1.5% agarose gel with suitable size markers (1000 bp) to determine fragment size.
57. Add 100 mL wash buffer (WB) 1 (RIPA Buffer, add 0.1% SDS just before use).a.Incubate the tubes for 4 min at 40 rpm on a rotator in the cold room (+4 C). b.Repeat one more time.58.Add 100 mL of WB 2 (add 0.1% SDS just before use).a.Incubate the tubes for 4 min at 40 rpm on a rotator in the cold room.b.Repeat one more time.59.Add 100 mL of WB 3 (add 0.1% SDS just before use).a.Incubate the tubes for 4 min at 40 rpm on a rotator in the cold room.b.Repeat one more time.60.Add 100 mL of TE buffer and incubate tubes for 4 min at 40 rpm on a rotator in the cold room.61.Before removing the TE buffer, transfer the mixture to a new tube and place it on the magnet for 1 min remove the TE buffer.62. Add 100 mL of TE buffer and incubate tubes for 4 min at 40 rpm on a rotator in the cold room and remove the TE (10 mM Tris-HCl pH 8.1 and 1 mM EDTA).

Figure 2 .
Figure 2. Results of RT-qPCR analysis of two genomic regions across samples of SAT and OVAT (A: actin gene, NC: Human negative control (commercial), S: Sat, V: OVAT)

Figure 3 .
Figure 3. Results of RT-qPCR analysis of ADIPOQ gene across samples of SAT and OVAT (ADI: ADIPOQ gene, NC: Human negative control (commercial), S: Sat, V: OVAT) Store at 4 C for up to 12 months.
Incubate the beads for 1 min on ice.a.Place them on the magnet for 1 min.b.Remove the buffer by gentle pipetting.Note: To avoid beads trapped in the lid, snap-spin the tube in a mini-centrifuge before putting the tube on the magnet.47.Repeat two times steps 45 and 46.48.Resuspend the beads in 103 volume of RIPA buffer (100 mL) containing 0.1% SDS.49.Add 1 mL of 2.5 mg H3K36me3 antibody to each 1.5 mL DNA low-binding tube.Note: Do gentle pipetting and do not vortexing.If requires, snap-spin the tube in a minicentrifuge.
: Calculate the amount of beads you need based on the number of ChIPs.45.Add 2.53 volumes (25 mL) of RIPA buffer (0.1% SDS) (containing PIC, NaBu, and PMSF) with gentle pipetting.46.50.Incubate the tubes (beads + antibody) at 40 rpm on a rotator in the COLD ROOM (+4 C) for 2 h.

Table 1 .
qPCR reaction master mix for positive region

Table 2 .
qPCR reaction master mix negative region